Identification of an intramolecular cross-link in troponin C after cross-linking the troponin complex with 1,3-difluoro-4,6-dinitrobenzene.

The troponin complex isolated from rabbit skeletal muscle was cross-linked with 1,3-difluoro-4,6-dinitrobenzene, subjected to tryptic hydrolysis, and the labelled peptides were separated. One peptide was purified to homogeneity; it proved to be peptide Gly(89)-Arg(100) of troponin C on the basis of amino acid composition and N-terminal analysis. The absorption spectrum of this peptide between 300 and 500 nm was very similar to that of 1-(S-cysteinyl)-5-(N epsilon-lysyl)-2,4-dinitrobenzene, which could only be due to cross-link formation between Lys-90 and Cys-98. This finding is interpreted in terms of the proposed tertiary structure of troponin C.

[Enzymatic digestibility of peptides containing tert-butyltryptophan residues (author's transl)]. / Untersuchungen über den enzymatischen Abbau von tert-Butyltryptophan-haltigen Peptiden.

The effects of tert-butylation of tryptophan residues in peptide chains on the enzymatic digestibility were investigated. It was shown that replacement of tryptophan in position 9 of Corticotropin-(1--19)-nonadecapeptidamide by a mono- or tri-tert-butylated tryptophan enhances the resistance of the resulting analogue against proteolytic enzymes.

Preparation of a target specific fraction controlling the proliferation of granulocytes. The abolishment of the specificity by glutathion.

A low molecular weight fraction (GI-3) was prepared from peripheral horse blood leukocytes. This fraction inhibits the proliferation of bone marrow cells, the effect is dose dependent. The proliferation of thymocytes and HeLa cells is not affected by GI-3. This specificity was abolished when glutathion was applied together with GI-3; the 3H-TdR uptake of thymocytes in vitro was completely inhibited, however, slight or no effect was observed on bone marrow and HeLa cells, respectively.

Some structural features of rabbit muscle aldolase as derived from its limited proteolysis.

The peptides released during the limited tryptic proteolysis of rabbit muscle aldolase (Biszku et al., 1973) were located in the primary structure. The pattern of peptide liberation, peptide bond splitting and activity decrease in compatible with two structural models for the truncated tetrameric product, named aldolase-T. According to the more probable model aldolase-T has the structure A+A+B++B++. Subunits B++ are deprived of the segments comprising residues 1-27, 42-71 and 306-364 of the intact enzyme and are inactive. The fragment comprising residues 28-41 is non-covalently attached to these subunits. Subunits A+ are depleted only of peptides 1-27 and 324-332 and retain 70% activity. In these subunits the fragment comprising residue 333-364 remains non-covalently bound. The molecular weights of the truncated subunits, determined with polyacrylamide-gel electrophoresis in the presence of sodium dodecylsulfate support the above conclusions. Aldolase-T can be reversibly denatured at pH 2 or in 4 M urea. The recovery of enzymatic activity after decreasing urea or acid concentration indicates the non-covalent rebinding of fragment 333-364. This fragment is named the "T-peptide" of trypsin-treated aldolase. It is suggested that segments 1-27 and 324-364 are not necessary for the renaturation process. Since aldolase-T is a tetramer it seems that large parts of the N- and C-terminal regions of the enzyme are not involved in the intersubunit interactions. The C-terminal region of aldolase, starting around residue 324, appears to be necessary to the structure of the active site. In contrast to this, the N-terminal region up to residue 27 and probably to residue 60, is not part of the active center.

Purification of an endopeptide to homogenity and the verification of its selective inhibitory action on myeloid cell proliferation.

The purification of an endogenous regulatory peptide to homogeneity which selectively inhibits the proliferation of normal and leukaemic myeloid cells is described. Leucocytes isolated from calf spleen or horse blood were homogenized, extracted by acetone, chloroform and distilled water, ultrafiltrated by Amicon Diaflo XM 50 and PM 10 membranes. The lyophilized filtrate was chromatographed on Sephadex G-15 and G-10 columns. Fractions were tested for the selective inhibition of myeloid proliferation by 3H-TdR incorporation and capillary colony formation. The active fractions were submitted to paper electrophoresis at pH 6.5 and 1.9, and the peptides were re-tested. Finally a ninhydrin negative and chlor-tolidine positive oligopeptide was identified as granulocyte-specific inhibitor, effective at 0.2 to 3.0 pmol/ml MED value in vitro. The peptide is negatively charged at pH 6.5, its electrophoretic mobility as compared to aspartic acid is -0.60. The peptide has no charge at pH 1.9, the mobility related to epsilon-DNP lysine is 0.26. Details of the structure analysis of the inhibitory endopeptide is described in our next paper.

Identification of a fluorescent dye-containing peptide of glyceraldehyde-3-phosphate dehydrogenase.

The NAD-induced local conformational changes in the fluorescent dye-binding region of muscle glyceraldehyde-3-phosphate dehydrogenase were studied (Ovádi et al., 1982). We have isolated a dominant peptide containing the fluorescent dye from the tryptic digest of labelled dehydrogenase, and identified this labelled amino acid residue. The data indicate that fluorescein isothiocyanate can react rather specifically with tyrosyl residue 91, which is not associated with the catalytic function of the enzyme. Tyrosyl-91 modified by the fluorescent dye does not interact directly with any part of the NAD bound at the active site of glyceraldehyde-3-phosphate dehydrogenase.

Comparative screening for thyroid disorders in old age in areas of iodine deficiency, long-term iodine prophylaxis and abundant iodine intake.

OBJECTIVE: To investigate the effect of varying amounts of iodine intake on the prevalence of thyroid dysfunction, autoimmunity and goitre in old age. DESIGN: The first screening study where elderly subjects with varying amounts of iodine supply but from the same geographical and ethnographical region (Carpathian basin) were compared, and all hormone measurements and ultrasonography were performed by the same laboratory or person. PATIENTS: Nursing home residents were screened for thyroid disorders from: (A) an iodine-deficient area, Northern Hungary (n = 119; median age 81 years; median iodine excretion (MIE) 0.065 mumol/mmol creatinine (equivalent to 72 micrograms/g creatinine); (B) an area of obligatory iodinated salt prophylaxis since the 1950s, Slovakia (n = 135; median age 81 years, MIE 0.090 mumol/mmol creatinine (equivalent to 100 micrograms/g creatinine)) and (C) an abundant iodine intake area, Eastern Hungary (n = 92; median age 78 years; MIE 0.462 mumol/mmol creatinine (equivalent to 513 micrograms/g creatinine)). MEASUREMENTS: TSH, T4, free T4, T3, thyroglobulin (Tg), antibodies to Tg (AbTg) and to thyroid peroxidase (AbTPO), iodine excretion, ultrasonography of the thyroid gland. RESULTS: In regions A, B, and C, the prevalence of unsuspected clinical hypothyroidism was 0.8%, 1.5% and 7.6% (P = 0.006), with all cases except one being antibody positive (Ab+). The occurrence of subclinical hypothyroidism was 4.2% in region A, 10.4% in region B and 23.9% in region C (P < 0.001), but only 3 of 22 cases with subclinical hypothyroidism from region C were Ab+. The overall prevalence of Ab positivity (either antiTg+ or antiTPO+) was similar in the three regions (A, 19.3%; B, 24.4%; C, 22.8%). The occurrence of hyperthyroidism (clinical plus subclinical) was 3.4% in region A, 3.0% in region B and 0% in region C (not significant). The rate of elevated Tg levels was similar in the three regions. The prevalence of goitre was 39.4%, 16.4% and 12.2% (P < 0.001), respectively in regions A, B and C. In euthyroid subjects the mean ultrasonographically determined thyroid volume was 21.9 ml in region A, 13.6 ml in region B and 15.1 ml in region C (ANOVA F = 5.76; P = 0.0038). There was no significant difference in the occurrence of cases with hypoechogenic echotexture of the thyroid gland. CONCLUSIONS: The screening for hypothyroidism in nursing home residents living in iodine-rich regions is justified by the high prevalence of unsuspected clinical hypothyroidism. The high prevalence of antibody positivity in old age is independent of the iodine supply, but iodine supply has a determining role in the development of autoimmune hypothyroidism in the aged. Most cases of subclinical hypothyroidism in iodine-rich regions are not of autoimmune origin. In old age, hypoechogenic texture of the thyroid gland is not predictive of thyroid dysfunction.

Is an amphiphilic region responsible for the haemolytic activity of Bacillus thuringiensis toxin?

The amino acid sequence of the 27 kDa protein responsible for the haemolytic activity of Bacillus thuringiensis subsp. israelensis toxin has been analysed by secondary structure prediction, helical wheel/net diagrams and molecular mechanics calculations. We found that segment 116-126 presumably forms a strongly amphiphilic alpha-helix. This is supported by the findings that the synthesized segment 116-126 (a) has a significant alpha-helical content in water, and (b) displays an in vitro haemolytic activity comparable to that of bee venom peptide melittin. As segment 116-126 is present in the haemolyzing, but not present in the non-haemolyzing proteins from B. thuringiensis toxins, we suggest that this segment is responsible for the lytic potential of the B. thuringiensis subsp. israelensis protein.